In order to elucidate the metabolic functions of NAD- and NADP- requiring two malic enzymes in Escherichia coli W, various factors controlling the enzyme syntheses and metabolic flow of C4 acids in the cell were studied. The NAD-requiring malic enzyme (NAD-enzyme) [EC 1.1.1.38 and 39] was repressed by glucose and induced by malate. The addition of glucose to a malate-containing medium caused a suppression of the enzyme level, but it was overcome by increasing the concentration of malate. On the other hand, the NADP-requiring malic enzyme (NADP-enzyme) [EC 1.1.1.40] was repressed not only by glucose, but also by glycerol, lactate, and acetate in decreasing order of magnitude. Since the reactions to yield acetyl-CoA from the above three compounds via pyruvate are actually irreversible in vivo, acetyl-CoA or acetate was presumed to have a close relation to the repression of the enzyme. However, the concentration of acetyl-CoA in the cell had no correlation with the level of NADP-enzyme and increasing concentrations of acetate did not exert any marked repression. Although pyruvate exhibited no appreciable repression by itself, combination of pyruvate and acetate exerted a marked repression of the enzyme. In addition, this was found to be the case also with a pyruvate dehydrogenase [EC 1.2.4.1]-less mutant of E. coli. Therefore, the effect of pyruvate was presumed not to be due to conversion of pyruvate into acetyl-CoA or acetate. These results indicate that NAD-enzyme undergoes an induction by malate and NADP-enzyme a concerted repression by acetate and pyruvate, besides the repression of both enzymes by glucose. In resting cells of the bacterium grown on the malate medium, the radioactivity was incorporated into fatty acids from 2, 3-14C-succinate but not from 1, 4-14C-succinate. These results support the view that malic enzyme(s) but not malate cleavage enzyme* is responsible for the formation of acetyl-CoA from malate. From these lines of evidence, together with allosteric properties of the two enzymes, the metabolic functions of these enzymes were postulated as follows: NAD-enzyme plays a role in the catabolism of malate, controlling the level of C4-dicarboxylic acids. In contrast, NADP-enzyme functions in the supply of pyruvate for the syntheses of various amino acids such as alanine, valine, and leucine, and of acetyl-CoA for the operation of the tricarboxylic acid cycle as well as for the syntheses of lipids or for the reductive biosyntheses by generating NADPH2